The family of the proteolytic mammalian subtilisin-like proprotein convertases (SPC or PC) is homologous with bacterial subtilisins and yeast Kex2p. To date seven distinct members of the SPC family have been identified, including furin, PC1 (also known as PC3), PC2, PACE4, PC4, PC5 (also known as PC6), PC7 (LPC, PC8, or SPC7), each of which exhibits unique tissue distribution.
All SPCs are multidomain enzymes, composed of an amino-terminal propeptide, a subtilisin-like catalytic domain, a middle domain and a unique carboxy-terminus composed of one or more domains. The pro-, catalytic and middle domains are essential and sufficient for catalytic activity. The carboxy-terminal domains are thought to contain the information for correct targeting to, and concentration in, the compartment of the secretory pathway in which the enzymes function.
These proteins have been implicated in the endoproteolytic maturation processing of inactive precursor proteins including hormones, growth factors, receptors, viral and bacterial proteins, and plasma proteins such as albumin, von Willebrand Factor (VWF), factor VII, factor IX, and factor X at single, paired or multiple basic consensus sites (Nakayama, Biochem J., 1997; 327:625-35).
The SPC member furin, also termed PACE (paired basic amino acid cleavage enzyme) is ubiquitously expressed in all mammalian tissues and cell lines and is capable of processing a wide range of bioactive precursor proteins in the secretory pathway, including also hormones, growth factors, receptors, viral and bacterial proteins, and plasma proteins. It is a calcium-dependent serine endoprotease structurally arranged into several domains, namely a signal peptide, propeptide, catalytic domain, middle domain, (also termed homo-B or P-domain), the C-terminally located cysteine-rich domain, transmembrane domain and the cytoplasmic tail. The furin protease cleavage site comprises a recognition sequence which is characterized by the amino acid sequence Arg-X-Lys/Arg-Arg (Hosaka et al., J Biol Chem. 1991; 266:12127-30).
Intact furin is incorporated into the membrane system of the Golgi apparatus and there it is functionally active (Bresnahan et al., J Cell Biol. 1990; 111:2851-9). Upon transit of the newly synthesized furin precursor from the endoplasmic reticulum to the Golgi compartment, the propeptide is autocatalytically removed in a two step processing event (Anderson et al., EMBO J. 1997; 16:1508-18).
Furin also cycles between the trans-Golgi network and the cell surface via endosomal vesicles, thereby processing both precursor proteins during their transport through the constitutive secretory pathway as well as molecules entering the endocytic pathway. The cellular distribution of furin to the processing compartments is directed by defined structural features within its cytoplasmic tail (Teuchert et al., J Biol Chem. 1999; 274:8199-07).
Since an overexpression of the protease negatively affects the growth of continuously growing cell cultures, solutions have been sought to reduce the toxic influence of furin on the cells. The C-terminal domains have been found to be dispensable for the functional activity of furin and a truncated form of the over-expressed native furin of 75-80 kD could be detected in the cell supernatant as secreted protein (Wise et al., PNAS. 1990; 87:9378-82). This naturally secreted truncated furin is also known as “shed furin” (Vidricaire et al., Biochem Biophys Res Comm. 1993; 195:1011-8; Plaimauer et al., Biochem J. 2001; 354:689-95) and is cleaved N-terminally of the transmembrane portion (Vey et al., J Cell Biol. 1994; 127:1829-42).
Furin proteins truncated by genetic engineering, in which the encoding part of the transmembrane and cytoplasmatic domains has been deleted have been described for example for amino acids Δ714-794 (Leduc et al., J Biol Chem. 1992; 267:14304-8; Molloy et al., J Biol Chem. 1992; 267:16396-402) and for amino acids Δ716-794 (“Sol-PACE”, Wasley et al., J Biol Chem. 1993; 268:8458-65; Rehemtulla and Kaufman, Blood. 1992; 79:2349-55) and for amino acids Δ705-794 (Hatsuzawa et al., J Biol. Chem. 1992; 267:16094-9). Furin mutants additionally comprising a deletion of the cystein-rich region have also been described (Hatsuzawa et al., J Biochem. 1992; 101:296-301; Creemers et al., J Biol Chem. 1993; 268:21826-34).
For biotechnological use in vitro as well as in vivo applications of SPCs are conceivable, including an application within the framework of a therapeutic treatment. For such applications, human furin or truncated furin may be more suitable than endopeptidases of non-human origin.
Furin or truncated furin may be applicable in the commercial production of all sorts of biologically active substances (e.g., other enzymes) if processing is a production step therein. For example the University of Leuven holds patents for the application of furin in the industrial production of biomedical relevant products (U.S. Pat. Nos. 5,989,856, 5,935,815, and 6,274,365).
Another example is the processing of pro-VWF. Actually the endoproteolytic activity of furin and its selectivity for basic amino acids has first been determined in experiments with pro-VWF. Pro-VWF consists of a propolypeptide with 741 amino acids and mature VWF with 2050 amino acids (Verweij et al., EMBO J. 1986; 5:1839-47) and is processed into its mature form by endogenously occurring furin (Wise et al., PNAS 1990; 87:9378-82; Van de Ven et al., Mol Biol Rep. 1990; 14:265-75; Rehemtulla and Kaufman, Blood. 1992; 79:2349-55). Because in the downstream process recombinant VWF (rVWF) consists of up to 50-70% of pro-rVWF, not fully maturated pro-rVWF has then to be further processed in vitro. Maturation of pro-rVWF can be achieved by addition of CHO cell supernatant containing unpurified furin to supernatant of unpurified pro-rVWF. However, due to low pro-rVWF and furin concentrations, this maturation process can last up to several days and is not very reproducible. Thus a purified furin or furin derivative would be preferable for the maturation process.
Despite the potential widespread use of furin or truncated furin in the maturation of proteins surprisingly there are only a few disclosures of methods for the purification of furin.
Recombinant truncated mouse furin has been purified only by a factor of 7 with a yield of 27% by purification with an anion exchange membrane followed by Mono Q and Superose 12 columns (Cameron et al., J Biol Chem. 2000; 275:36741-9).
Hatsuzawa et al. (J Biol Chem. 1992; 267:16094-9) achieved a 100-fold purification of truncated mouse furin in CHO cells with a relatively small yield of about 10% by using ammonium sulfate fractionation, TOYOPEARL® AF-Blue batchwise fractionation and DEAE-TOYOPEARL® chromatography. The same method was also used by Nakayama et al. as published in Methods Enzymol. 1994; 244:167-75.
All these methods, however, due to the use of several consecutive steps are either relatively time consuming, have a low purification degree or a low yield of furin. Such furin purification methods are not useful for a large scale industrial process where furin is only needed on top of the protein to be prepared. Thus there is a great need in the art for a fast furin purification process in combination with a high purification degree and a high yield. It was an inventive task of the present invention to develop a simple single column purification step for human recombinant furin or truncated furin with a high yield and sufficient purification degree to allow maturation of pro-proteins such as for example pro-rVWF. Furthermore, a subsequent additional purification step should be developed to obtain an essentially pure furin or truncated furin.